Geographical distance between locations plays a crucial role in establishing healthcare accessibility in Two-Step Floating Catchment Area (2SFCA) models. In this article, we analyze three sets of distances: a straight-line distance and two network-based distances to determine how the distance calculation affects the models. We empirically examine the distances across several 2SFCA models and several input parameters. We find that the accessibilities derived from network-based distances correlate with each other, whereas the accessibility derived from straight-line distance does not correlate with them. The study is based on the data from the South Moravian region.